Project Summary Obesity predisposes humans to diabetes and cardiovascular disease and is a universal threat to health. Fat storage is dynamically controlled by orchestrated hormonal, neural and metabolic signals between the brain and periphery. Proopiomelanocortin (POMC) neurons that synthesize melanocortin peptides are a primary integrative site for these diverse signals related to energy homeostasis. In the past funding cycles, we made significant progress towards explaining how Pomc transcription is restricted to a subset of hypothalamic neurons in the arcuate nucleus. A modular locus comprised of two evolutionarily distinct enhancers, nPE1 and nPE2, directs neuron-specific Pomc expression. Targeted deletion of the individual or combination of enhancers revealed that they act synergistically during hypothalamic development and additively in adult life to maintain sufficiently robust Pomc transcription to avoid obesity. A bioinformatic analysis of core nucleotide sequence motifs within the enhancers combined with anatomic and functional interrogation of candidate homeobox transcription factors (TFs) in animal models identified a major contribution of Isl1 and Nkx2.1 in directing the unique temporal and spatial patterns of Pomc expression in the arcuate nucleus. The Rax TF also contributes to the identity of POMC neurons developmentally, but acts indirectly upstream of Isl1. Although necessary, these factors alone are not sufficient to fully account for the complexities of neuronal Pomc regulation. Translating Ribosome Affinity Purification (TRAP) seq of POMC neurons has identified additional, highly differentially expressed TF genes that are putatively involved in the control of Pomc expression. Therefore, we propose the following specific aims for this project renewal: 1) Uncover the complete genetic program controlling hypothalamic Pomc expression through the functional characterization of candidate TFs that define the early identity of POMC neurons and their maintenance throughout the entire lifetime, using mouse molecular genetics and human pluripotential stem cells (hPSCs) that can be differentiated into POMC neurons in vitro; 2) Determine the cis-acting code that defines hypothalamic Pomc expression through the in vivo functional analysis of the entire set of neuron-specific enhancers, their critical binding motifs and local insulators to assemble a fully functional transcriptional locus; and 3) Dissect the physiological significance of individual enhancers and critical motifs involved in the hormonal regulation of Pomc expression by leptin and in response to altered dietary conditions and caloric demands. These studies will provide fundamental knowledge about a gene essential for regulating body mass and possibly identify novel genetic or signaling pathways that can be exploited for therapeutic purposes.